appeared as individual, uniformly stained bacilli (Determine 1?1AA and ?andB).B). diverse, but the most common manifestations are septicemia (50% of cases), pneumonia, and abscesses in internal organs.3 Mortality in Thailand is 40%, rising to AKR1C3-IN-1 90% in those with severe sepsis. Rapid diagnosis and administration of effective antimicrobial therapy is usually life saving, because is usually inherently resistant to a range of antibiotic classes, and patients require ceftazidime or a carbapenem drug.1,3,4 Laboratory diagnosis is culture-based, which takes at least 48 hours from sample receipt to confirmed identification. Molecular methods including real-time polymerase chain reaction (PCR) and loop-isothermal amplification have been described, which can be applied to DNA extracted directly from the clinical sample, and these can provide a more quick diagnosis but have a lower diagnostic sensitivity than culture.5,6 Our clinical research laboratory in Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand has used an in-house immunofluorescence microscopy assay (IFA) for the rapid detection of in clinical specimens since 1993.7 This uses a fluorescein isothiocyanate (FITC)-labeled rabbit polyclonal antibody (Pab) against formalin-killed exopolysaccharide,14 and the aim of this study was to re-evaluate the IFA in the laboratory and clinical settings after replacing the Pab with this Mab and replacing FITC with a photo-stable dye. The Mab-IFA was developed as an indirect assay. The primary detection antibody was unlabelled Mab 4B11 (IgG2b subclass) specific to exopolysaccharide,14 and the secondary antibody was Alexa Fluor 488 conjugated-goat anti-mouse immunoglobulin G (IgG) (Molecular Probes, Carlsbad, CA). Mab was prepared from culture supernatant of hybridroma clone 4B11, as explained previously.14 The Mab-IFA was optimized for ease of use by preparing a single mixture of AKR1C3-IN-1 primary and secondary antibody, which was added to the slide in a single step. The Mab-IFA detection reagent contained 5 g/mL of Mab and 20 g/mL of secondary antibody in phosphate-buffered AKR1C3-IN-1 saline (PBS). The limit of detection was defined using a 10-fold dilution series ranging from 21010 to 20 colony-forming models (CFU)/mL of K96243. Ten microliters of each bacterial dilution was mixed with an equal volume of Mab-IFA and incubated at room heat for 5 min before observing for the presence of AKR1C3-IN-1 green fluorescent bacteria using a fluorescent microscope at 1,000 magnification (Olympus BH-2, Tokyo, Japan). appeared as individual, uniformly stained bacilli (Physique 1?1AA and ?andB).B). The limit of detection of the assay, defined as the lowest quantity of bacteria that gave a positive result for Mab-IFA, was 2103 CFU/mL. Open in a separate window Physique 1. Fluorescent microscopy of stained with Mab-IFA reagent. The bacteria shown were from laboratory cultures on Columbia agar (A) or LB broth (B), or from clinical samples (urine [C], pus [D], or sputum [E]) from patients with melioidosis. The atypical appearance of the bacterial morphology including bacterial elongation (D and E) and swollen cells (C) was not uncommon. The assay sensitivity of the Mab-IFA was defined using 20 clinical isolates. The Mab-IFA assay specificity was defined by screening 160 microorganisms representing a wide range of species. These were 20 Gram-positive bacteria (, unknown species of -hemolytic , spp. ), 136 Gram-negative bacteria (spp. , , ), , , ), spp. , , , spp. , , spp. , , , [1), spp. , [1), spp. , , , , , , , spp. , , , , , , TEAD4 , , , , , serovar Paratyphi A , serovar Typhi , , , ), and 4 fungi (spp. , ). Microorganisms were sub-cultured on Columbia agar and incubated overnight at 37C in air flow. Fastidious bacteria were sub-cultured on chocolate agar and incubated overnight at 37C in AKR1C3-IN-1 5% CO2. The assay sensitivity was 100% (20 of 20 positive),.